Interactions among molecules such as proteins are fundamental to cell biology. Protein binding to a wide variety of cellular components, including proteins, nucleic acids, carbohydrates, and lipids, has been recognized as an important drug target due to its integral nature within signal transduction and biological pathways. Such binding can be correlated to a variety of intracellular events, including protein expression, the availability of an active state of a protein, and, directly or indirectly, to protein catalytic activity. For instance, in the cytoplasm the protein kinase MAPK, when complexed with MEK1, is inactive. Upon activation, MEK1 and MAPK dissociate, leading to free, activated MAPK. Detection of the activated MAPK by virtue of its ability to bind to a binding domain in a target substrate indicates the presence of the active enzyme, and is indirectly related to the MAPK activity of phosphorylating substrates.
Current methods for analyzing cellular molecular binding events, such as two-hybrid systems and variants thereof, substrate complementation systems, immunoprecipitation assays, in vivo incorporation of radiolabeled moieties, and the use of antibodies specific for a given modification (such as phosphorylation), suffer from numerous drawbacks. Such drawbacks include the need to construct two or more chimeric proteins; the inability to monitor biochemical events in live, intact cells or in fixed cells; the requirement for considerable time to conduct the assays; and the need for specialized and expensive equipment. Thus, improved reagents and methods for detecting and measuring specific binding events are needed.
A very significant improvement would be a flexible design for reagents and assays that can be used to detect molecular binding events that occur within living cells. Such reagents would preferably comprise a single chimeric protein, and would be applicable to monitoring molecular binding events in live and fixed end point cell preparations as well as to making kinetic measurements of the binding events in cells. Such reagents would preferably possess detectable signals that permit easy detection of molecular binding events of interest, and also provide the ability to combine the molecular binding event assay with other cell-based assays.